ONE-STEP Tagging: a Versatile Method for Rapid Site-Specific Integration by Simultaneous Reagent Delivery

Site-specific integration of DNA sequences into the genome is an important tool in fundamental research, synthetic biology and cell therapeutic applications. It can be used for protein tagging to investigate expression, localisation, and interactions as well as for expression of transgenes either under endogenous regulatory elements or at consistent safe harbour loci. Here we develop and optimise a simple and effective method for site specific integration in a single step that combines CRISPR-Cas9 mediated homology directed repair using single stranded oligonucleotide templates with the site-specific recombinase Bxb1 to allow large cargos to be integrated at any location in the genome. Our technology requires off the shelf Cas9 and oligonucleotide reagents combined with a set of cargo plasmids that are universal to any integration site. We demonstrate the methods adaptability by tagging at multiple sites and in multiple cell types including induced pluripotent stem cells and primary T cells. We show that our method can integrate large (up to 14 kb) cargos and that it is possible to simultaneously tag two genes or edit two sites with combination of integration and Cas9-mediated knockouts or other HDR events. ### Competing Interest Statement AB is a founder of and consultant for Ensocell therapeutics.